Hepatoblasts and method of isolating same

ABSTRACT

This invention relates to methods of isolating hepatoblasts utilizing panning techniques and fluorescence activated cell sorting. This invention further relates to isolated hepatoblasts and to a method of treating liver dysfunction as well as to methods of forming artificial livers.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application is a Continuation-In-Part of Application Ser.No. 07/741,128 filed Aug. 7, 1991, entitled PROLIFERATION OF HEPATOCYTEPRECURSORS.

FIELD OF THE INVENTION

[0002] This invention relates to methods for isolating hepatoblasts andto said isolated hepatoblasts. The isolated hepatoblasts of theinvention comprise liver stem cells (pluripotent precursors) andcommitted progenitors (precursors with only one fate) for eitherhepatocytes or bile duct cells. The isolated hepatoblasts of theinvention may be used to treat liver dysfunction and for artificiallivers, gene therapy, drug testing and vaccine production. In addition,the isolated hepatoblasts of the invention may be used for research,therapeutic and commercial purposes which require the use of populationsof functional liver cells.

[0003] Unlike mature liver cells, the hepatoblasts of the inventiongenerate daughter cells that can mature through the liver lineage andoffer the entire range of liver functions, many of which arelineage-position specific. Further, the hepatoblasts of the inventionhave a greater capacity for proliferation and long-term viability thando mature liver cells. As a result, the hepatoblasts of the inventionare better for research, therapeutic and commercial uses than matureliver cells.

BACKGROUND OF THE INVENTION

[0004] Stem cells and early progenitors have long been known to exist inrapidly proliferating adult tissues such as bone marrow, gut andepidermis, but have only recently been thought to exist in quiescenttissues such as adult liver, an organ characterized by a long cellularlife span. The ability of stem cells to self-replicate and producedaughter cells with multiple fates distinguishes them from committedprogenitors. In contrast, committed progenitors produce daughter cellswith only one fate in terms of cell type, and these cells undergo agradual maturation process wherein differentiated functions appear in alineage-position-dependent process.

[0005] In adult organisms, stem cells in somatic tissues produce alineage of daughter cells that undergo a unidirectional, terminaldifferentiation process. In all well-characterized lineage systems, suchas hemopoiesis, gut and epidermis, stem cells have been identified byempirical assays in which the stem cells were shown to be capable ofproducing the full range of descendants. To date, no molecular markersare known which uniquely identify stem cells as a general class ofcells, and no molecular mechanisms are known which result in theconversion of cells from self-replication and pluripotency to acommitment to differentiation and a single fate.

[0006] The structural and functional units of the hepatic-parenchyma isthe acinus, which is organized like a wheel around two distinct vascularbeds. Six sets of portal triads, each with a portal venule, a hepaticarteriole and a bile duct, form the periphery, and the central veinforms the hub. The parenchyma, which comprises the “spokes” of thewheel, consists of plates of cells lined on both sides by thefenestrated sinusoidal endothelium. Blood flows from the portal venulesand hepatic arterioles at the portal triads, through sinusoids whichalign plates of parenchyma, to the terminal hepatic venules, the centralvein. Hepatocytes display marked morphologic, biochemical and functionalheterogeneity based on their acinar location (see Gebbardt, Pharmac.Ther., Vol. 53, pp. 275-354 (1990)).

[0007] Comparatively, periportal parenchymal cells are small in size,midacinar cells are intermediate in size and pericentral cells arelargest in size. There are acinar-position-dependent variations in themorphology of mitochondria, endoplasmic reticulum and glycogen granules.Of critical importance is that the diploid parenchymal cells and thosewith greatest growth potential are located periportally. In parallel,tissue-specific gene expression is acinar-position-dependent leading tothe hypothesis that the expression of genes is maturation-dependent (seeSigal et al., Amer. J. Physiol., Vol. 263, pp. G139-G148 (1993)).

[0008] It is currently believed that the liver is a stem cell andlineage system which has several parallels to the gut, skin andhemopoietic systems (see Sigal et al., Amer. J. Physiol., Vol. 263, pp.G139-G148 (1993); Sigal et al. In Extracellular Matrix, Zem and Reed,eds., Marcel Decker, NY., pp. 507-537 (1993); and Broil et al., LiverBiology and Pathobiology, Arias et al., 3d eds., Raven Press, NY (1994in press)). As such, it is expected that there are progenitor cellpopulations in the livers of all or most ages of animals. A lineagemodel of the liver would clarify why researches have been unable to growadult, mature liver cells in culture for more than a few rounds ofdivision, have observed only a few divisions of mature, adult livercells when injected in vivo into liver or into ectopic sites, and havehad limited success in establishing artificial livers with adult livercells. These impasses are of considerable concern in the use of isolatedliver cells for liver transplantation, artificial livers, gene therapyand other therapeutic and commercial uses.

[0009] The success of the above-listed procedures requires the use ofhepatic progenitor cells (hepatoblasts) which are found in a highproportion of liver cells in early embryonic livers and in small numberslocated periportally in adult livers. Because it is desirable to isolatesuch hepatoblasts, a need has arisen to develop a method of successfullyisolating said hepatoblasts. The inventors have identified markers anddeveloped a method for isolating hepatoblasts from the livers of animalsat any age. The methods of the invention have been developed usingembryonic and neonatal livers from rats, however, the method of theinvention offers a systematic approach to isolating hepatoblasts fromany age from any species.

[0010] The methods of the invention have been developed with embryoniclivers in which there are significant numbers of pluripotent liver cells(liver stem cells) and committed progenitors (cells with a single fateto become either hepatocytes or bile duct cells). The onset ofdifferentiation of rat parenchymal cells of the liver occurs by thetenth day of gestation. By this stage, parenchymal cells (epithelial orepitheloid cells) are morphologically homogeneous and consist of smallcells with scant cytoplasm and, therefore, high nuclear to cytoplasmicratios, with undifferentiated, pale, nuclei and a few intercellularadhesions. Most liver parenchymal cells at this stage are considered tobe bipotent for bile duct cells and hepatocytes. Although they express,usually weakly, some liver-specific functions known to be activated veryearly in development, such as albumin and α-fetoprotein (AFP), they donot express adult-specific markers such as glycogen, urea-cycle enzymesor major urinary protein (MUP). Only a few islands of fetal cells arepositive for BDS₇, a bile duct cell-specific marker, and none arepositive for HES₆, a hepatocyte-specific marker (see Germain et al.,Cancer Research, Vol. 48., pp. 4909-4918 (1988)). The hepatoblasts withscant cytoplasm and often ovoid-shaped nuclei comprise several cellpopulations including pluripotent liver stem cells and committedprogenitors, each having only one fate for either bile duct cells orhepatocytes.

[0011] By the fifteenth day of gestation, hepatoblasts increasingly arecomprised of the committed progenitors that differentiate along eitherthe bile duct or the hepatocytic lineage. Their maturation is denoted bychanges in morphology (increasing size, increasing numbers ofcytoplasmic organelles and vacuoles, heterogeneous nuclear morphologiesand an increase in pigmented granules), which can be distinguishedreadily by flow cytometric parameters. “Forward scatter” measures cellsize. “Side scatter” measures cellular complexity or granularity, whichis affected by the numbers of cellular organelles. Autofluorescence isdependent upon lipofuscins and other pigments that increase withmaturation.

[0012] Accompanying the morphological changes are step-wise orsequential changes in expression of types of cytokeratins, varioussurface antigens and tissue-specific genes. Whereas the earlyhepatoblasts which include liver stem cells intensely express AFP andweakly express albumin, committed progenitors destined to becomehepatocytes form cords of cells that lose their AFP expression, expressincreasingly high levels of albumin and gradually acquirehepatocyte-specific markers such as glycogen and urea cycle enzymes.Cells destined to become intrahepatic bile duct cells arise fromseemingly identical hepatoblasts and retain expression of AFP, losealbumin expression and acquire cytokeratin 19 (CK 19). Initially, astring of pearl-like cells is present around the large vascular branchesclose to the liver hilium. Over the ensuing days, similar structuresappear throughout the liver. BDS₇-positive cells rapidly enlarge andbecome more numerous with increasing developmental age. Gradually,lumina form within the structures, and by the eighteenth day ofgestation, bile ductular structures are morphologically identifiable.

[0013] In order to understand liver development and the sequentialchanges in the expression of liver-specific genes with maturation, it isnecessary to study the hepatoblasts directly. However, the study ofhepatoblasts is hindered by the difficulty in isolating them since theyalways constitute a small portion, less than 10%, of the cell typeswithin the liver in embryonic, neonatal, and adult life. In the embryo,the liver is the site for both hepatopoiesis (formation of liver cells)and hemopoiesis (formation of blood cells). Hempoietic cells migratefrom the yolk sac into the liver during the twelfth day of gestation.Subsequently, hemopoiesis, particularly erythropoiesis, rapidly becomesone of the most prominent functions of the fetal liver with hemopoieticcells comprising 50% or more of the liver mass. In neonates, themajority of the liver cells are either hemopoietic cells or mature livercells (hepatocytes or bile duct cells). As a result, sequential changesin parenchymal functions in intact liver are difficult to interpretbecause the data are confounded by the changing hemopoieticcontributions. For example, it has been demonstrated that a transientdecrease in parenchymal functions at day eighteen of gestation is duenot to a decrease in hepatic cells or in their expression of thesegenes, but occurs because it is the peak of erythropoiesis, when most ofthe liver consists of erythroid cells. Hemopoiesis in the liver declinesrapidly after birth as it transfers to the bone marrow, the site ofhemopoiesis in the adult. Nevertheless, isolation of hepatoblasts inadult liver remains problematic, since they comprise a very smallpercentage of hepatic cells.

[0014] Because hepatoblasts can generate all developmental stages ofliver cells and, therefore, offer the entire range of liver-specificfunctions encoded by genes activated and expressed in early to latestages of differentiation, have much greater growth potential thanmature liver cells, have greater proliferative potential and offer cellswith greater ability for transfection with appropriate genes (i.e.,greater capacity for gene therapy), it is desirable to isolatehepatoblasts (as opposed to mature liver cells).

[0015] Currently available methods for isolation of hepatoblasts requirethe use of fractionation methods for cell size or cell density which areinadequate for separating the hemopoietic from the hepatopoieticprecursors, require the use of cells surviving specific enzymetreatments such as pronase digestion (which have been proven to alsokill hepatoblast subpopulations) or require the use of selectionprotocols in culture in which enrichment of the cells of interest aredependent upon differential attachment to the substratum or differentialgrowth in specific culture media. Hence, currently available isolationmethods have proven very inefficient. Moreover, identification of theparenchymal cell precursors is dependent upon assays forparenchymal-specific functions. Further, hepatoblasts dedifferentiateunder most culture conditions and thereby come undetectable, or thereare such a high proportion of non-relevant cells (e.g., mesenchymalcells) that the functions of interest are swamped out by those of thecontaminant cell populations. In addition, dissociated liver cellsreadily from large aggregates via a calcium- and temperature-dependentglycoprotein-mediated process. In order to disaggregate the liver cells,it is necessary to utilize mechanical methods including vigorouspipetting and aspiration through a syringe, methods which are usuallyinsufficient to achieve single cell suspensions and which can result indramatically reduced viability of the cells. Hence it is desirable todevelop a method of isolating fetal hepatoblasts which method maintainsthe hepatoblasts as a single cell suspension, does not result in cellaggregation, and is applicable to all ages.

[0016] It is therefore an object of this invention to provide methods ofisolating hepatoblasts.

[0017] It is a further object of this invention to provide isolatedhepatoblasts.

[0018] It is another object of this invention to provide a method ofutilizing isolated hepatoblasts to treat liver dysfunction.

[0019] It is a still further object of this invention to provide methodsof forming artificial livers utilizing isolated hepatoblasts.

SUMMARY OF THE INVENTION

[0020] This invention relates to isolated hepatoblasts and to methods ofisolating hepatoblasts utilizing panning techniques and flow cytometry(fluorescence activated cell sorting) on cell suspensions of livercells. Dissociated liver cells are panned and fluorescence activatedcell sorted utilizing antibodies so as to greatly reduce the numbers ofcontaminating cell types, such as hemopoietic cells in embryonic liveror mature liver cells in adults. The cells that do not adhere to thepanning dishes are negatively sorted using multiple antibodies to thecontaminant cell types which leads to a cell population highly enrichedfor immature hepatic cell types, and then segregated into distinctsubcategories of immature hepatic cell types by multiparametricfluorescence activated cell sorting. This invention is further directedto the use of isolated hepatoblasts for the treatment of liverdysfunction and for the production of artificial livers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above brief description, as well as further objects andfeatures of the present invention, will be more fully understood byreference to the following detailed description of the presentlypreferred, albeit illustrative, embodiments of the present inventionwhen taken in conjunction with the accompanying drawings wherein:

[0022]FIG. 1 represents cells from day 14 gestation livers stained formonoclonal antibodies 374.3 and OX-43, followed by FITC and PE-labeledsecond antibodies. Panel A is a two color density plot showing 5populations designated R1-5 in an ungated sample. R1 and R2 are cellpopulations positive for OX-43, while R3-5 are negative for this marker.Panel B is a biparametric dot plot of FL2 versus SSC showing the gatingparameters used to separate OX-43⁺ from OX-43⁻ cells. The insert showsthe negative control. Panel C is a 3D plot of FL1 versus FL2 of OX-43⁻cells showing three distinct cell populations, R3-5;

[0023]FIG. 2, panel A is a Western blot of total protein from sortedcells showing the presence of albumin containing cells exclusively inthe OX43⁻ population. Panels B and C show indirect immunofluorescencefor AFP on OX-43⁻ (B) and OX-43⁺ (C) cells;

[0024]FIG. 3 represents cells from R3-5 which were sorted after gatingout all OX-43⁺ cells and total RNA prepared by the guanidiniumisothiocyanate method. The Northern blot demonstrates expression ofalbumin in R4, while serglycin is expressed by R3 cells;

[0025]FIG. 4 represents cells which were gated to separate populationspositive and negative to OX-43 and then further separated to 5populations based on their fluorescence on biparametric density plots ofFL1 versus FL2. Freshly sorted and cytospun cells were stained formorphology by Diff-Quik staining kit. Original magnification—100×;

[0026]FIG. 5 represents a population highly enriched for fetal liverparenchymal cells which was obtained by FACS (R4 cells after exclusionof all OX-43⁻) and 5×10⁴ cells/cm² plated on type I collagen coateddishes in a serum free, hormonally defined medium. Panel A is a phasemicrograph showing a typical epithelial colony and very few mesenchymalcells after 4 days in culture (original magnification—5×). Panel B is anindirect in situ immunofluorescence showing incorporation of BrdU in thenuclei of about 25% of the cultured parenchymal cells after 24 hours inculture (original magnification—5×). Panel C is a phase micrograph ofpanel B;

[0027]FIG. 6 represents a flow diagram of hepatoblast enrichmentutilizing a method of the invention;

[0028]FIG. 7 panel A represents phase contrast microscopy and panel Brepresents immunofluorescence for AFP of hepatoblasts at gestation day15. AFP positive cells ranged in morphology from small cells with ovalnuclei and scant cytoplasm that were only slightly larger than thehemopoietic cells to cells with larger amounts of vacuolated cytoplasm.Negative controls consisted of cells stained with rabbit IgG as aprimary antibody;

[0029]FIG. 8 represents Northern blot analysis of total RNA (5 μg/lane)from freshly isolated fetal liver cells before and after panning andhybridized with cDNAs encoding α-fetoprotein and albumin. Lane 1 showsfreshly isolated fetal liver cells. Lane 2 shows cell preparation afterpanning 2× with anti-rat RBC antibody. Also shown are blots for 18S,used as an internal control for total RNA loading;

[0030]FIG. 9 represents biparametric analysis of fetal rat liver cellspresented as side scatter (SSC), a measure of cytoplasmic complexity,versus log fluorescence for OX-43 and OX-44. Panel A shows unstainedcells; panel B shows the cells immediately following isolation (originalsuspension); and panel C shows the cells after final panning. The vastmajority of the cells immediately after isolation were agranular andpositive for the markers (R1 cell population). With enrichment, thepopulation of granular cells (SSC>50 A.U.) which were negative for theOX43/OX44 markers (R3 cell population) increased. Sorting for thispopulation revealed that 75% were positive for AFP. The demarcationbetween positive and negative is higher for the granular than theagranular populations due to greater autofluorescence of the granularcells;

[0031]FIG. 10 represents day 15 gestation cells enriched forhepatoblasts by panning out RBCs cultured for 5 days on type IV collagenin serum-free hormonally defined medium. The cells exhibited typicalepithelial morphology including formation of bile canaliculi.Surrounding epithelial cells are fibroblast-like cells. Bar=25μ; and

[0032]FIG. 11 represents small epithelial islands showing positivestaining for albumin by in situ immunofluorescence after 16 days inculture. The fibroblast-like cells surrounding them are negative for thepresence of albumin. Bar=100μ.

DETAILED DESCRIPTION OF THE INVENTION

[0033] This invention relates to isolated hepatoblasts and to methods ofisolating hepatoblasts from dissociated liver cells utilizing panningtechniques and fluorescence activated cell sorting. The isolatedhepatoblasts of the invention can be used to treat liver dysfunction, toproduce artificial livers, in the study of liver functions, in genetherapy, in drug testing and in vaccine production.

[0034] Livers are dissociated by enzymatic digestion, avoiding enzymessuch as pronase that adversely affect hepatoblasts, and then kept insolutions which are chilled and which contain chelating agents such asEGTA, which results in cells that can be sustained as single cells.Dissociated liver cells are then panned with antibodies to greatlyreduce the numbers of contaminating cell types (hemopoietic cells,including red blood cells, endothelial cells and other mesenchymal cellsin embryonic and neonatal liver, and mature liver cells, hepatocytes,bile duct cells, endothelial cells and other mesenchymal cells in adultliver). Panning alone, although rapid, is inefficient and does not yieldvery pure cell populations. However, it is used to rapidly reduce thenumber of non-hepatoblast cells. The cells that do not adhere to thepanning dishes are then segregated by fluorescence activated cellsorting, a technology with very high accuracy and efficiency. Thecombination of the rapid panning methodology with the accuracy of thefluorescence activated cell sorting results in highly purified cellpopulations with good viability.

[0035] In embryonic and neonatal livers, the contaminant cell typesreduced through panning protocols are erythroid, myeloid and otherhemopoietic cell types and endothelia (mesenchymal cell types). Thepanning steps lead to a cell population enriched for immature hepaticcell types. In adult livers, the contaminant cell types are maturehepatocytes, bile duct cells, endothelia and some hemopoietic cellpopulations.

[0036] Panned cells are also sorted for multiple markers thatdistinguish distinct subcategories of hepatic precursor cellpopulations. The markers identified are (a) the extent of granularity asmeasured by side scatter on fluorescence activated cell sorting, whereinmore immature cell populations are more agranular, and increasinggranularity correlates with increasing maturity; (b) the extent ofautofluorescence, wherein increasing autofluorescence correlates withincreasing maturity; and/or (c) the expression of a hepatic cell marker(such as the oval cell marker OC.3, which is detected by monoclonalantibody 374.3).

[0037] Liver cells which do not express hemopoietic or endothelial cellantigens recognized by monoclonal antibodies OX-43 and/or OX-44 (whichrecognize myeloid cells and endothelia) and which do not expressantigens recognized by a monoclonal antibody to an erythroid antigencomprise the hepatoblasts of the invention. The hepatoblasts of theinvention include three categories of immature liver cells:

[0038] (1) More granular cells, which are OC.3⁺, are committed bile ductprecursors. These cells are also AFP⁺, albumin⁺ and CK 19⁺.

[0039] (2) More granular cells, which are OC.3⁻. are committedhepatocyte precursors. These cells are also AFP⁺, albumin⁺⁺⁺, and CK19⁻.

[0040] (3) Agranular cells, which are OC.3⁺, are very immature hepaticprecursors. These cells are also AFP⁺⁺⁺, albumin⁺ and CK 19⁻

[0041] This invention is further directed to the use of hepatoblastsisolated by the methods of the invention. The isolated hepatoblasts ofthe invention can be used for to treat liver dysfunction. For example,hepatoblasts can be injected into the body, such as into the liver orinto an ectopic site. Whole liver transplantation, which requires costlyand dangerous major surgery, can be replaced by a minor surgicalprocedure which introduces hepatoblasts in vivo either into the livervia the portal vein or at an ectopic site such as the spleen. Inaddition, hepatoblasts can be used in bioreactors or in cultureapparatus to form artificial livers. Further, hepatoblasts can be usedin gene therapy, drug testing, vaccine production and any research,commercial or therapeutic purpose which requires liver cells of varyingextents of maturity.

EXAMPLE I

[0042] Fischer 344 rats with known durations of pregnancy were obtainedfrom Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) and maintained inthe animal facility of the Albert Einstein College of Medicine, Bronx,N.Y. on a standard rat chow diet with 12 hour light cycles. Byconvention, the first day of gestation is defined as day 0. Use ofanimals was in accordance with the NIH Policy on the care and use oflaboratory animals and was approved by the Animal Care and Use Committeeof the Albert Einstein College of Medicine.

[0043] In order to isolate fetal liver cells, pregnant rats at thefourteenth day of gestation were euthanized with ether and the embryoswere removed intact and placed into ice cold CA⁺²-free Hank's BalancedSalt Solution containing 0.04% DNAse, 0.8 mM MgCl₂, 20 mM HEPES, pH 7.3(HBSS). Livers were then dissected from the fetuses and placed intofresh ice-cold HBSS. After all tissues were collected and non-hepatictissue removed, HBSS-5 mM EGTA was added to a final EGTA concentrationof 1 mM. The livers were moved to a 50 ml conical centrifuge tube bypipette, gently triturated 6 to 8 times to partially disaggregate thetissue and then centrifuged at 400 g for 5 minutes at 4° C. Allsubsequent centrifugation steps were performed at the same settings. Thesupernatant was removed and the pellet of cells and tissue wasresuspended in 50 ml 0.6% Collagenase D (Boehringer Mannheim,Indianapolis, Ind.) in HBSS containing 1 mM CaCl₂, gently triturated andthen stirred at 37° C. for 15 minutes in an Erlenmeyer flask. Thedispersed cells were pooled, suspended in HBSS containing 1 mM EGTA andfiltered through a 46 μm tissue collector (Bellco Glass, Inc., Vineland,N.Y.). The cell suspension was centrifuged and the cells wereresuspended in HBSS supplemented with MEM amino acids, MEM vitamins, MEMnon-essential amino acids, insulin (10 μ/ml), iron-saturated transferrin(10 μg/ml), free fatty acids (7.6 mEq/L, as described by Chessebeuf etal., 1984, Nu-Chek-Prep, Elysian, Minn.), trace elements, albumin (0.1%,fraction V, fatty acid free, Miles Inc., Kankakee, Ill.), myo-inositol(0.5 mM) and gentamicin (10 μg/ml, Gibco BRL, Grand Island, N.Y.)(HBSS-MEM). Cell number and viability were determined by hemacytometerand trypan blue exclusion.

[0044] In order to remove erythroid cells, panning dishes were preparedaccording to the procedure of Wysocki and Sato (1978) using a rabbitanti-rat RBC IgG (Rockland Inc., Gilbertsville, Pa.). Antibodies (0.5mg/dish) diluted in 9 ml of 0.05 M Tris pH 9.5 were poured on 100 mm²bacteriological polystyrene petri dishes (Falcon, Lincoln Park, N.J.).The dishes were swirled to evenly coat the surface and incubated at roomtemperature for 40 minutes. The coated dishes were washed four timeswith PBS and once with HBSS containing 0.1% BSA prior to use.

[0045] Three milliliters of the cell suspension containing up to 3×10⁷cells were incubated at 4° C. for 10 minutes in the dishes coated withthe rabbit anti-rat RBC IgG. The non-adherent cells were removed byaspiration and the plates were washed three times with HBSS-0. 1%BSA-0.2 mM EGTA and centrifuged. The cell pellet was resuspended inHBSS-MEM and RBC panning was repeated. Following the second RBC panningcell number and viability were determined again.

[0046] The cells recovered after RBC panning were then labeled insuspension by incubating with mouse monoclonal antibody OX-43 (1/200=15μg/ml, MCA 276, Bioproducts for Science, Indianapolis, Ind.) andmonoclonal antibody 374.3 (1/500-1/750, a gift of R. Faris and D. Hixon,Brown University, Providence, R.I.) simultaneously at 4° C. for 40minutes. OX-43 recognizes an antigen on endothelial cells, asubpopulation of macrophages and erythroid cells (see Barclay,Immunology, Vol. 42, pp. 593-600 (1981) and Robinson et al., Immunology,Vol. 57, pp. 231-237 (1986)) and 374.3 recognizes oval cells, bile ductcells and hemopoietic cells (see Hixon et al., Pathology: LiverCarcinogenesis, pp. 65-77 (1990)). Second antibodies were PE-conjugatedanti-mouse IgG, heavy chain specific (Southern Biotechnology Inc., AL.)and FITC-conjugated anti-mouse IgM, heavy chain specific (Sigma ChemicalCo., St. Louis, Mo.). Negative controls included cells without label andcells labeled with mouse isotype controls.

[0047] Cells before and after sorting were maintained at 4° C. and inHBSS-MEM. After completion of the antibody labeling, propidium iodide atfinal concentration of 10 μg/ml was added to each of the sample tubes.Fluorescence Activated Cell Sorting was performed with a BectonDickinson FACSTAR^(plus) (San Jose, Calif.) using a 4W argon laser with60 mW of power and a 100 μm nozzle. Fluorescent emission at 488 nmexcitation was collected after passing through a 530/30 nm band passfilter for FITC and 585/42 nm for PE. Fluorescence measurements wereperformed using logarithmic amplification on biparametric plots of FL1(FITC) vs FL2 (PE). Cells were considered positive when fluorescence wasgreater than 95% of the negative control cells.

[0048] For measurement of physical characteristics of the cells,FACSTAR^(plus) parameters were FSC gain 8 and SSC gain 8. These settingsallowed all cells to be visualized on scale. HBSS was utilized as sheathfluid. For analysis, a minimum of 10,000 events were measured. List modedata were acquired and analyzed using LysisII software. Dead cells weregated out using propidium iodide fluorescence histograms on unlabeledcells.

[0049] For determination of positivity to a single antibody dot plots offluorescence versus side scatter were used. Density plots FL1 versus FL2were used to select populations with respect to expression of bothantigens. A sort enhancement module was utilized for non-rectangulargating and use of multiparametric gating to select populations ofinterest.

[0050] Shorted cells from day fourteen of gestation from all populationswere plated in a serum-free, hormonally-defined medium with αXMEM as thebasal medium to which the following components were added: insulin (10μg/ml); EGF (0.01 μg/ml, Upstate Biotechnology, Lake Placid, N.Y.);growth hormone (10 μU/ml); prolactin (20 μU/ml); Triiodothyronine (10⁻⁷M); dexamethasone (10⁻⁷ M); iron saturated transferrin (10 μg/ml);folinic acid (10⁻⁸ M, Gibco BRL, Grand Island, N.Y.), free fatty acidmixture (7.6 mEq/L, as described by Chessebeuf et al., 1984,Nu-Chek-Prep, Elysian, Minn.); putrescine (0.02 μg/ml); hypoxanthine(0.24 μg/ml); thymidine (0.07 μg/ml); bovine albumin (0.1%, fraction V,fatty acid free, Miles Inc. Kankakee, Ill.); trace elements; CuSO₄.5H₂O(0.0000025 mg/l), FeSO₄.7H₂O (0.8 mg/l) MnSO₄.7H₂O (0.0000024 mg/l),(NH₄)₆MO₇O₂₄.H₂O (0.0012 mg/l), NiCl₂.6H₂O (0.000012 mg/l), NH₄VO₃(0.000058 mg/l), H₂SeO₃ (0.00039 mg/l); Hepes (31 mM) and Gentamicin (10μg/ml, Gibco BRL, Grand Island, N.Y.). Reagents were supplied by SigmaChemical Company, St. Louis, Mo., unless otherwise specified. The traceelement mix was a gift from Dr. I. Lemishka, Princeton University, NJ.

[0051] Culture dishes as well as cytospins of various cell suspensionswere fixed with ice-cold ethanol or acetone. After blocking with PBScontaining 1% BSA for 30 minutes at room temperature, the fixed cellswere studied by indirect immunofluorescence using the following primaryantibodies: polyclonal rabbit-anti-rat albumin (United StatesBiochemical Corporation, Cleveland, Ohio), rabbit-anti-mouse AEPantiserum (ICN Biomedical, In., Costa Mesa, Calif.), monoclonalmouse-anti-human cytokeratin 19 (Amersham Life Science, ArlingtonHeights, Ill.), polyclonal rabbit-anti-human IGF II receptor (a gift ofDr. Michael Czech, University of Worchester, MA), mouse monoclonalanti-rat-Thy-1 (OX-7, Bioproducts for Science, Indianapolis, Ind.),monoclonal mouse-anti-desmin (Boehringer Mannheim, Indianapolis, Ind.),and 258.26, a monoclonal mouse-anti-rat antibody identifying postnatalhepatocytes as well as some fetal liver parenchymal cells (a gift ofDrs. R. Fans and D. Hixon, Brown University, RI). Second antibodiesincluded species, specific Rhodamine conjugated antibodies correspondingto the primary antibodies. Negative controls consisted of cells stainedwith mouse or rabbit IgG or mouse isotype controls. Freshly isolatedadult hepatocytes were used as positive controls for albumin staining.Gamma-glutamyltranspeptidase (GGT) was assayed by immunochemistry onethanol fixed cells using the method described by Rutenberg et al., J.Hist. Cyt., Vol. 17, pp. 517-526 (1969).

[0052] In order to perform Northern blot analysis for the presence ofspecific mRNA, total RNA was extracted from sorted cells using theguanidinium isothiocyanate method, as described by Chomcznyski et al.,Anal. Biochem., Vol. 162, pp. 156-159 (1987)). RNA samples were resolvedby electrophoresis through 1% agarose formaldehyde gels in3-(N-morpholino)-propanesulfonic acid buffer (see Maniatis et al.,Molecular Cloning: A Laboratory Manual, pp. 191-193 (1982)). The RNA wasthen transferred to Gene Screen (New England Nuclear, Boston. Mass.),and the filters were prehybridized and hybridized with the appropriateprobes. The cDNA clones complementary to specific mRNAs wereradioactively labeled by primer extension with 32P dCTP as described byFeinberg et al., Anal. Biochem., Vol. 137, pp. 266-267 (1984). The cDNAsused in hybridization were rat albumin (a gift of Dr. Zern, JeffersonUniversity, Philadelphia, Pa.), and mouse α-fetoprotein, (Dr. Tighlman,Princeton, NJ), GGT (obtained from Dr. M. Manson, MRC Medical ResearchCouncil, Surrey, UK) and PG19. Autoradiograms were scanned with aQuantimat densitometer (Model 920; Manufacturer's Cambridge Instrument).The data for each of the genes was normalized to that for the commongene 18S (J. Darnell, Rockefeller University, New York, N.Y.).

[0053] In order to perform Western blot analysis, total protein samplesfrom various sorted cells were loaded on a 10% polyacrylamide minigel.Loading was normalized for equal cell numbers, 100,000 cells per slot.Electrophoresis followed by electroblotting to nitrocellulose membranes(Schleicher and Schuell, Keene, N.H.) was performed. The blots wereblocked overnight in 2% dry milk solution at 4° C. and assayed foralbumin using a rabbit-anti-rat albumin antiserum diluted 1:800 in theblocking solution for 1 hour at room temperature, followed by a one hourincubation with horseradish-peroxidase-conjugated anti-rabbit IgG(Amersham Life Science, Arlington Heights, Ill.) diluted 1:50 inblocking solution. Detection was achieved by incubation of blots withECL-chemiluminescence kit reagents (Amersham Life Science, ArlingtonHeights, Ill.) for 1 minute and subsequent autoradiography.

[0054] Forty-eight well plates were coated with type I collagenextracted from rat tail tendon as described by Reid, Methods inMolecular Biology, The Humana Press, Inc., Vol. 5, pp. 237-276 (1990).Sorted cells at densities between 50,000 to 100,000 cells/cm² wereplated per well. Following an overnight attachment period, the mediumwith the non-adhering cells was gently removed and replaced by freshmedium. A complete medium change was performed every 24 hours. The cellswere cultured at 37° C. in a fully humidified atmosphere containing 5%CO₂ and were observed daily. After four days in culture, cells werefixed with ice-cold ethanol and stained in situ by Immunofluorescencefor albumin, AFP, CK 19 and IGF II receptor and by immunochemistry forGGT, as described below.

[0055] Livers from fourteenth day gestation embryos isolated by theEGTA-collagenase digestion yielded single cell suspensions and anegligible number of cell aggregates. Cellular viability was greaterthan 95% as determined by exclusion of trypan blue. Cell yield was2.62±0.31×10⁶ cells per liver. The original cell suspension wassubjected to two steps of immunoadherence (“panning”) using rabbitanti-rat RBC IgG coated polystyrene dishes. Cellular recovery aftercompletion of two panning steps was 51% (±8%), but varied somewhat withdifferent lots of antibodies.

[0056] The cells recovered after RBC-panning were stained in suspensionwith a mixture of two antibodies: an antibody raised against “ovalcells” (monoclonal antibody 374.3) and a commercially available antibodyknown to recognize endothelial, as well as some erythroid and myeloidcells in the rat (monoclonal antibody OX-43). Following incubation withthe proper FITC and PE labeled second antibodies, cells were analyzedfor their fluorescence patterns. As shown in FIG. 1, panel A, whenfluorescence intensities for both antigens were plotted against eachother, five distinct populations, referred to as R1 through R5, wereobserved. With minor variations in the percentage of each population,the distribution of cells to form the five populations was extremelyreproducible. The small differences could be explained by variations inthe percent recovery of cells after RBC panning.

[0057] Initial analyses of sorted cells by immunofluorescence revealedthe presence of albumin and AFP positive cells in one of the OX-43positive cell populations (R2). These larger and more complex cellscomprised approximately 5-10% of cells in this gate. However, whenfreshly sorted R2 cells were viewed under the epi-fluorescentmicroscope, these larger cells appeared to be negative for OX-43 (no PElabeling). The parenchymal cells in the liver have a significant degreeof autofluorescence, which increases with maturation of the liver, inparallel to the increase in cellular complexity, as measured by the sidescatter parameter on the FACS. It was therefore postulated that it isdue to this phenomenon that some parenchymal cells appear in the regionof the OX-43-positive cells, although not expressing the antigen. Topursue this hypothesis, positivity to OX-43 was determined accurately onside scatter (cellular granularity) versus PE fluorescence, as measuredon the FL2 scale (FIG. 1, panel B), and OX-43-positive and negativecells were sorted and characterized. To determine the accuracy of thesorts, post-sort acquisitions of the sorted cells were performed usingthe same instrument settings. Typical post-sort purity (i.e., percentageof cells from a shorted population that appeared in the same region whenanalyzed again after the sort) was >90%.

[0058] Sorted cells from both OX-43 positive and negative gates wereassayed for expression of liver specific genes by Western blot analysisand by indirect immunofluorescence. As shown in FIG. 2, panel A, therewas a minimal amount of albumin in the OX-43-positive cell fraction,detected by Western blotting, as compared with the OX-43-negative cells.No AFP positive cells could be shown by indirect immuno-fluorescence oncytospins of sorted OX-43-positive cells, as opposed to 30% ofOX-43-negative cells expressing the fetal liver marker (see FIG. 2,panels B and C). It was concluded that at day 14 of gestation, all fetalliver parenchymal cells are OX-43-negative. Therefore, in order toachieve “cleaner” gates, OX-43-positive and negative cells wereseparated on a SSC versus FL2 plot and studied separately.

[0059] When OX-43 positive cells were electronically gated out and theremaining cells viewed on a FL1 versus FL2 plot, three distinctpopulations were readily detected (see FIG. 1, panel C), correspondingto R3-5 in the ungated cell suspension. All of the cells in R3 were374.3-positive whereas 30% of the cells in R4 were positive for thatmarker. R5 cells did not express OC.3. Expression of variousliver-specific and other genes was studied on sorted cells from R3-5.The results are summarized in Table 1, below. TABLE 1 Characterizationof sorted cells by immunofluorescence and by histochemistry R1 R2 R3 R4R5 Albumin neg neg  1% pos 75-80% pos neg AFP neg neg  2%. pos 70% posneg GGT neg neg  1% pos 75% neg IGF-IIr 20%  1%  2% 85% neg CK 19 negneg  2-3% neg neg Desmin <1%+  1-2% +++ neg neg <1% + 258.26 neg neg negneg neg Thy-1  2% 10% 75% 10%  5%

[0060] About 2-3% of R3 cells (less than 0.2% of the tota ungated cellsuspension) were intensely stained for albumin and AFP. They alsoexpressed GGT and CK 19, markers of the bile duct lineage. However themajority of the cells appeared to be small, blast-like cells, and didnot express liver specific genes but expressed classical hemopoieticmarkers such as Thy-1 and serglycin (see Table 1 and FIGS. 3 and 4).Most of the liver parenchymal cells were found in the R4 gate (see Table1 and FIG. 3). The vast majority of the cells expressed albumin, AFP andGGT, all markers of fetal liver parenchyma. No hemopoietic or fatstoring cell markers were detected in that gate. The cell populationdesignated R5 is a heterogeneous one (see FIG. 4), comprising mainly twocell types: (1) cells that morphologically appear to be normoblasts; and(2) simple small cells that did not express parenchymal liver genes. Theratio between these two cell types varied somewhat and was dependent onthe efficiency of the RBC panning.

[0061] When all of the OX-43 negative cells were gated out, two distinctpopulations were observed on an FL1/FL2 plot. As expected, noparenchymal liver markers were detected in these cells. A few of R2cells intensely stained with the antibody against desmin, anintermediate filament usually expressed in fat storing cells.Morphologically, most of R2 cells appeared to be early erythroidprecursors (see FIG. 4), while 10% of them expressed Thy-1. In the R1gate were two morphologically distinct cell types (see FIG. 4). Themajority were small, blast-like and did not express any of the markerstested. The others, about 20% of the cells in this gate, were largercells with a pale cytoplasm and expressed the receptor for IGF-II. Veryfew cells from R1 stained for Thy-1.

[0062] Sorted cells from all 5 populations were cultured for 4 days todetermine in vitro fates. When plated at high density under theconditions described, R4 cells yielded clusters of epithelial cellssurrounded by very few scattered stromal cells (see FIG. 5A and Table 2below). TABLE 2 Characterization of R4 cells after 4 days in cultureMarker Epithelial Cells Stromal Cells Albumin + neg AFP ± neg GGT ++ negCK 19 + (30%) neg 258.26 neg neg IGF IIr + (perinuclear staining) +(perinuclear staining)

[0063] Cell division was clearly evident both in the epithelial as wellas the stromal components of the culture. On the second day of theculture 25±5% of the epithelial cells showed incorporation ofbromo-deoxy-uridine (BrdU) following a one hour incubation with a mediumcontaining BrdU (see FIGS. 5A and B). When RBC-panned but not sorted day14 gestation cells were plated under similar conditions, they survivedfor at least 10 days (data now shown). However, cultures of sorted R4cells deteriorated quickly. The epithelial cells lost their classicalpolygonal shape and elongated, similarly to what is seen in primarycultures of adult hepatocyte in the presence of serum. Moreover, whenstained in situ for albumin, AFP and GGT, cultured R4 cells exhibited agradual decline in these liver-specific genes, whereas RBC-panned day 14gestation cells maintained their gene expression under similarconditions (data not shown). IGF-II receptor remained clearly detectedin the golgi of the cultured epithelial as well as the stromal cells.About 30% of the cultured R4 cells showed staining for CK 19, acytokeratin present in bile duct cells and not in adult hepatocytes.

[0064] When cells from all other four populations were plated under thesame conditions, only few scattered fibroblast-like cells (but notepithelial colonies) were observed. Despite the liver-parenchymalcharacteristics of some R3 cells, epithelial colonies from these cellscould not be obtained under similar plating conditions. This may havebeen due to low density of the epithelial cells in this gate. Thesecells aggregated in suspension, survived for about 48 hours and thendied. Coating the dishes with type I or type IV collagen, fibronectin orlaminin alone or in combination did not improve attachment or survivalof these cells (data now shown).

EXAMPLE II

[0065] Fisher 344 rats with known durations of pregnancy were obtainedfrom Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) and maintained inthe animal facility of the Albert Einstein College of Medicine, Bronx,N.Y. on a standard rat chow diet with 12 hour light cycles. Byconvention, the first day of gestation is defined as day 0. Use ofanimals was in accordance with the NIH Policy on the care and use oflaboratory animals and was approved by the Animal Care and use Committeeof the Albert Einstein College of Medicine.

[0066] Pregnant rats at the fifteenth day of gestation were euthanizedwith ether, and the embryos were delivered. Livers were then dissectedfrom the fetuses, weighed, placed into ice-cold, Ca⁺²-free Hank'sBalanced Saline Solution containing 0.8 mM MgCl₂, 20 mM HEPES, pH 7.3(HBSS), and gently agitated at room temperature for 1 minute. Afterremoval of non-hepatic tissue, livers were gently triturated and thenstirred at 37° C. for 10 to 15 minutes in an Erlenmeyer flask with 0.6%type IV collagenase (Sigma Chemical Co., Lot 11H6830, St. Louis, Mo.) inHBSS containing 1 mM CaCl₂ and 0.06% DNAse I (Boehringer Mannheim,Indianapolis, Ind.). At 5 minute intervals, tissue fragments wereallowed to sediment at 1 g. The supernatant was recovered and freshcollagenase solution added. The dispersed cells were pooled, suspendedin HBSS containing 5 mM EGTA and filtered through a 46 pm tissuecollector (Bellco Glass, Inc., Vineland, N.Y.) under 1 g. The resultantcell suspension was centrifuged at 4° C. for 5 minutes under 450 g. Thecell pellet was resuspended in HBSS containing 0.2 mM EGTA and 0.5% BSA(HBSS-EGTA-0.5% BSA), and the cell number was estimated with a CoulterCounter (Coulter Electronics, Inc., Hialeah, Fla.). Cell viability wasassessed by exclusion of 0.04% trypan blue, and an aliquot of thesuspension was centrifuged in a tared microfuge tube at 450 g for 5minutes.

[0067] In order to immunoadhere hemopoietic and endothelial cells ontoantibody-coated polystyrene dishes, panning dishes were preparedaccording to the procedure of Wysocki and Sato. The antibodies employedincluded rabbit anti-rat RBC IgG (Inter-cell Technologies, Inc.,Hopewell, N.J.) and goat IgG directed towards mouse whole IgG molecule(M-3014, Sigma, St. Louis, Mo.). Antibodies (0.5 mg/dish) diluted in 10ml of 0.05 M Tris pH 9.5 were poured on 100 mm² bacteriologicalpolystyrene petri dishes (Falcon, Lincoln Park, N.J.) to evenly coat thesurface and incubated at room temperature for 40 minutes. The coateddishes were washed four times with PBS and once with HBSS containing0.1% BSA prior to use.

[0068] Three milliliters of the cell suspension containing up to 3×10⁷cells were incubated at 4° C. for 10 minutes in the dishes coated withthe rabbit anti-rat RBC IgG. The supernatant containing non-adherentcells was removed by gentle aspiration while tilting and swirling,combined with three washes of 7 ml HBSS-EGTA-0.1% BSA, and centrifugedat 4° C. for 5 minutes under 450 g. Cells from two dishes were pooledand repanned with a fresh dish coated with rabbit anti-rat RBC IgG. Thenon-adherent cells were then removed as above and resuspended withHBSS-EGTA-0.5% BSA to a concentration of 1×10⁷/ml. The enrichedhepatoblasts were then incubated simultaneously at 4° C. for 40 minuteswith mouse monoclonal antibody OX-43 (15 μg/ml, MCA276, Serotec,Indianapolis, Ind.) and monoclonal antibody OX-44 (18 μg/ml, MCA371,Serotec, Indianapolis, Ind.). OX-43 recognizes an antigen onmacrophages, endothelial cells and red blood cells, and OX-44 recognizesthe membrane-glycoprotein CD53 that is present on all rat myeloid cellsas well as peripheral lymphoid cells, and is related to the humanleukocyte antigen CD37. After washing to remove excess antibody, cellswere panned at 4° C. for 10 minutes in a dish coated with the goatanti-mouse whole IgG antibody, and non-adherent cells were removed asdescribed above.

[0069] Next, cytospins of the various cell suspensions were fixed witheither ice-cold ethanol or alcohol, acetone and carbowax 1540 (Fix-Rite,Richard-Allan Medical Industries, Richland, Mich.). After blocking, thefixed cells were immunostained by indirect immunofluorescence or thebiotin/streptavidin method using β-galactosidase (BioGenex, San Ramon,Calif.) with rabbit anti-rat albumin IgG (USB Corp., Cleveland, Ohio) orrabbit anti-mouse AFP antiserum (ICN ImmunoBiologicals, Lisle, Ill.) asprimary antibodies. Negative controls consisted of cells stained withthe primary antibodies omitted. Positive controls for albumin stainingwere done with freshly isolated adult hepatocytes.

[0070] In order to perform Northern blot analysis, total RNA wasextracted from the cells before and after panning and from the cellsadherent to the panning dishes using the guanidinium isothiocyanatemethod. RNA samples were resolved by electrophoresis through 1% agaroseformaldehyde gels in 3-(N-morpholino)-propanesulfonic acid buffer, thentransferred to Gene Screen (New England Nuclear, Boston, Mass.), whichwas prehybridized, and then hybridized with the appropriate probes. ThecDNA clones complementary to specific mRNAs were radioactively labeledby primer extension with ³²p dCTP. The cDNAs used were rat albumin,mouse AFP and mouse 18S (J. Darnell, Rockefeller University, NY).Autoradiograms were scanned with a Quantimat densitometer (Model 920;Manufacturer's Cambridge Instrument). The data for each of the genes wasnormalized to that for the common gene 18S.

[0071] To perform FACS analysis and sorting for hemopoietic andendothelial cell markers at day 15 gestation, cell suspensions atvarious stages of enrichment were analyzed by flow cytometry in the FACSfacility of the Albert Einstein College of Medicine, Bronx, N.Y. Cellswere resuspended to 1×10⁷ cell/ml and incubated at 4° C. for 40 minuteswith OX-43 with and without OX-44, followed by FITC-conjugatedanti-mouse IgG (heavy chain specific, Southern Biotech, Birmingham,Ala.) at 4° C. for 40 minutes. Cells stained only with FITC-conjugatedanti-mouse IgG served as negative controls.

[0072] Flow cytometric analysis was performed on a Becton-DickinsonFACScan (San Jose, Calif.) with a 15mW air-cooled argon laser. Cellsorting was performed with a Becton Dickinson FACSTAR^(plus) (San Jose,Calif.) using a 4W argon laser with 60 mW of power and 100 μm nozzle. Inboth instances fluorescent emission at 488 nm excitation was collectedafter passing through a 530/30 nm band pass filter for FITC.Fluorescence measurements were performed using logarithmicamplification. Cells were considered positive when fluorescence wasgreater than 95% of the negative control cells. For measurement ofphysical characteristics of the cells, the detector value was E-1 forforward scatter (FSC) with mid-range amplification. For side scatter(SSC) the detector value was mid-range with an amplification of 1.Equivalent FACSTAR^(plus) parameters were FSC gain 4 and SSC gain 8.These settings allowed all cells to be visualized on scale. FSC and SSCgating were performed using linear amplifications dividing bothparameters into 256 arbitrary units (A.U.). For analysis, at least10,000 events were measured. List mode data were acquired and analyzedusing LysisII software. Cells before and after sorting were maintainedat 4° C. and in HBSS supplemented with insulin, transferrin, free fattyacids, trace elements, albumin, and gentamicin as detailed forsupplements added to the HDM.

[0073] Next, multiparametric flow cytometric analysis of hemopoietic andendothelial markers was performed with respect to the oval cell antigenOC.3. Isolated cells were labeled with a combination of OX-43 and OX-44(mouse IgGs) and monoclonal antibody 374.3 (mouse IgM, Hixson and Faris,Brown University, Providence, R.I.) followed by FITC-conjugated goatanti-mouse IGG (heavy chain specific, So Biotech, Birmingham, Ala.) andPE-conjugated goat anti-mouse IgM (heavy chain specific, So Biotech,Birmingham, Ala.). Cells stained only with FITC-conjugated anti-mouseIgG and PE-conjugated anti-mouse IgM served as negative controls. Cellswere evaluated both for extent of fluorescence for one of the probes andby side scatter, a measure of cellular complexity (extent of cytoplasmicorganelles).

[0074] Cells from day 15 gestation livers were panned against rat redblood cell antibody, and the epithelial-enriched cell suspension wasplated in a serum-free hormonally defined medium with AMEM as the basalmedium to which the following components were added: insulin (10 μg/ml);EGF (0.01 μg/ml), Upstate Biotechnology, Lake Placid, N.Y.); growthhormone (10 μU/ml); prolactin (20 mU/ml); glucagon (10 μg/ml);Triiodothyronine (10⁻⁷M); dexamethasone (10⁻⁷M); iron saturatedtransferrin (10 μg/ml); folinic acid (10⁻⁸M, Gibco BRL, Grand Island,N.Y.), free fatty acid mixture (0.76 mEq/l, a modification of the methoddescribed by Chessebeuf, Nu Check-Prep, Elysian Minn.); putrescine (0.02μg/ml); hypoxanthine (0.24 μg/ml); thymidine (0.07 μg/ml); bovinealbumin (0.1%, fraction V, fatty acid free, Miles Inc., Kankakee, Ill.);trace elements: CuSO₄.5H₂O (0.0000025 mg/1), FeSO₄.7H₂O (0.8 mg/1),MnSO₄.7H₂O (0.0000024 mg/1), (NH₄)₆Mo₇O₂₄.H₂O (0.0012 mg/1, NiCl₂.6H₂O(0.000012 mg/1), NH₄VO₃ (0.000058 mg/1), H₂SeO₃ (0.00039 mg/1); Hepes(31 mM) and Gentamicin (10 μg/ml, Gibco BRL, Grand Island, N.Y.).Reagents were supplied by Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified. The trace element mix was a gift from Dr. I.Lemishka, Princeton University, NJ.

[0075] Twenty-four well plates were coated with type IV collagenextracted from EHS tumors. Panned cells at densities between 12,500 and25,000 cells per cm² were plated per well and allowed to attach for fourto five hours after which the medium with the non-adhering cells weregently removed and replaced by fresh medium. Cells were cultured at 37°C. in a fully humidified atmosphere containing 5% CO₂ and were observeddaily for 5 to 16 days. A complete medium change was performed every 48hours.

[0076] At various time points after initiation of the culture, cellswere fixed with ice-cold ethanol and stained in situ by immunochemistryor by immunofluorescence for albumin and AFP.

[0077] The weight of the liver at the 15th day of gestation was 9.1±1.3mg. Collagenase treatment digested the liver completely, and onlyminimal particulate matter was excluded by the tissue sieve. The numberof cells obtained at this step was 1.07×10⁷/liver, and the weight of thedissociated cells was 8.6±1.1 mg/liver, 95% of the whole organ weight.The suspension consisted almost entirely of isolated single cells withoccasional small aggregates that increased in size and number in theabsence of EGTA and at temperatures greater than 4° C. Viability bytrypan blue exclusion was greater than 90%.

[0078] After each panning, phase contrast microscopy demonstrated thatthe adherent cells exhibited an erythroid morphology. Only rare cellswere positive for albumin by immunochemistry. After panning with therabbit anti-rat red blood cell antibody-coated dishes to remove redblood cells and then with the goat anti-mouse whole molecular IgGantibody-coated dishes to reduce the numbers of OX43/OX44⁺ cells, thenon-adherent cells constituted 29±5% and 16±4%, respectively, of thecell number of the freshly dispersed fetal liver (original suspension).Panning proved successful for liver tissue at all fetal and earlyneonatal ages, although the variation in hemopoietic constituents withdevelopmental age resulted in differing degrees of enrichment (data notshown). Also, the efficiency of the RBC panning procedure varied withthe antibody lot. With antibodies of poor efficiency for direct panning,however, indirect immunoadherence was successful for the cells labeledin suspension followed by panning with anti-rabbit IgG coated petridishes.

[0079] On phase contrast microscopy following liver dispersion thepredominant cell type was a small, red cell consistent in morphologywith that of an early erythroid cell. Also present were larger,vacuolated cells. hnmunocytochemistry demonstrated that the vastmajority of the vacuolated cells as well as occasional smaller,oval-shaped cells were strongly positive for albumin and AFP (see FIG.7). The proportions of albumin and AFP positive cells at various stagesof enrichment are shown in (see Table 3 below and FIG. 6). TABLE 3Characteristics of the E15 liver cellular suspension at various statesof enrichment Percent of cells positive in the Percent of cells Percentof cells Original positive after positive after Markers Suspension RBCPanning IgG Panning Albumin¹  3.2 ± 1.3  9.5 ± 1.2 14.8 ± 3.6Alpha-fetoprotein  2.5 ± 0.7  9.8 ± 0.9 14.9 ± 2.5 MoAb OX-43² 76.6 ±5.8 70.5 ± 6.1 ND MoAb OX-43/44² 87.9 ± 2.5 80.4 ± 3.9  69.0 ± 10.0 %cells remaining of 100 29 ± 5 16 ± 4 original suspension

[0080] Northern blot analysis for liver-specific genes (albumin and AFP)was done on cells before and after panning and is shown in FIG. 8. Thecells after panning were enriched up to 5-fold for AFP MRNA and 2-foldfor albumin mRNA, a finding indicative both of the success of thepanning procedures and of the high concentrations of hepatoblasts (asopposed to mature hepatocytes). Negligible levels of albumin and no AFPmRNA were evident in the cells adherent to the panning dishes.

[0081] To determine the efficiency with which hemopoietic andendothelial cells were removed, cells at various stages of enrichmentwere analyzed by flow cytometry for the presence of OX-43 whichrecognizes macrophages, endothelial cells and red blood cells and forthe presence of OX-44 which recognizes myeloid and peripheral lymphoidcells. The results are shown in FIG. 6 and in Table 3. The percentage ofcells positive for OX-43/OX-44 in the original cell suspension was87.9±2.5%. The combination of panning procedures with anti-rat RBC IgGand anti-mouse whole IgG antibodies removed 84% of the cells. Although69±10.0% of the non-adherent cells were still positive for the OX-43/44markers, the percentage of hepatoblasts was enriched dramatically(5-fold). Although additional panning could have reduced the OX-43/44⁺cell population even further, it was found that the cell numbers hadbeen reduced sufficiently by panning to enable the FAC sorting tocomplete the process of eliminating the OX-43/44⁺ cells.

[0082] When examined by flow cytometry, fetal liver cells constituted aheterogeneous population with respect to FSC, a measure of cell size,and SSC, a measure of cytoplasmic complexity. Cytologically, there was abroad range in cell size (5 to 15 μ by Coulter Counter, data not shown),but cell size was not found to be useful in separating hemopoietic fromparenchymal precursors. Rather, the populations were best segregatedusing SSC. The definition of granular versus agranular cells was madebased on a linear scale for side scatter using biparametric plots offluorescence versus side scatter. Based on the population profiles, 50A.U. usually demarcated the agranular from the granular cells.

[0083] Using SSC versus fluorescence, the fetal liver cells could beisolated into three populations: agranular cells (the R1 population),which were positive for the endothelial and/or myeloid markers(OX43/OX44), and agranular (R2) and granular (R3) cells negative for theOX43/OX44 markers (see FIG. 9). The demarcation between positive andnegative was higher for the granular than the agranular populations dueto greater autofluorescence of the granular cells. Analysis of thesorted FACS populations demonstrated that less than 1% and 3.0±0.7% ofthe cells in the R1 and R2 populations, respectively, were positive forAFP. However, 75.1±4.7% of the granular cells negative for the markers(R3) were positive for AFP by immunocytochemistry (see Table 4 below).TABLE 4 Characteristics of cell fractions on FACS R1 R2 R3 Fluorescencefor 276 positive negative negative and/or 371¹ Granularity (A.U.)²agranular agranular granular % AFP positive³ <1% 3.0 ± 0.78% 75.1 ± 4.7%

[0084] Double image analysis of the R1 cell population, the only oneanalyzed having OX-43/OX44⁺ cells, indicated extensive overlap ofOX-43/44 positive and OC.3 positive cells. The FACS pattern forOX-43/OX-44 was similar for all gestational ages except for a subtleincrease in the R1 (and concomitant decrease in the R3 population) withincreasing gestational age due to increasing hepatic erythropoiesis(data not shown). Analysis of the sorted cell population that waspositive for OX-43/44, regardless of expression of OC.3 or ofgranularity, revealed that morphologically most were hemopoieticprecursor cells and were negative for AFP. Of the granular, OX-43/44⁻cells (the R3 cell population), most of which were AFP⁺, approximately30% were OC.3⁺. A small population of cells (R2 in Table 4) that wereOX43/44⁻, agranular, and AFP⁺ have not been evaluated for OC.3expression.

[0085] Cell preparations from day 15 gestation enriched by panning forhepatoblasts were plated on type IV collagen-coated dishes and in theserum-free, hormonally defined medium as described. Within a day afterplating, the epithelial cells reaggregated and attached to the matrix assmall cell clusters. Plating efficiencies of up to 60% were obtained(data not shown). The cells were organized into islands of typicalparenchymal cells forming close cell-cell contacts and bile canaliculi,surrounded by non-epithelial, fibroblast-like cells (see FIG. 10). After4-5 days in culture the parenchymal cell components were graduallyovergrown by the non-parenchymal cells. However, residual clusters ofhepatoblasts remained positive for albumin and AFP for up to 16 days inculture, as assessed by n situ immunochemistry or immunofluorescence(see FIG. 11). In a few experiments in which glucagon was omitted fromthe culture medium, no noticeable morphological difference was observed,and the cells expressed albumin and AFP when stained in situ byimmunofluorescence or immunochemistry (data not shown). This observationis attributed to relative glucagon resistance of the fetal hepatoblasts.

[0086] The inventors have developed methods, incorporating panningtechnologies and multiparametric FAC sorting, which isolate cellpopulations highly enriched for liver parenchymal cell precursors. Themethods of this invention have been found by the inventors to beapplicable to the isolation of hepatic precursor cells from liver fromgestational age day 13 through the early neonatal period. The liverdispersion procedure described yields a population of predominantlysingle cells with greater than 90% viability, and at gestation day 15,95% of the whole organ weight is recovered. The panning proceduresremove up to 84% of the total cell number, and simultaneously enrich thehepatoblast population by 5-fold. The increase in theparenchymal-specific gene expression of albumin and AFP was illustratedby Northern blot analysis of the cells before and after panning, and theprocedure's specificity demonstrated by analysis of the cells adherentto the panning dishes. Similarly, the enrichment was confirmed by the invitro data in which there was a dramatic increase in the number of cellcolonies expressing albumin and AFP after panning compared to theoriginal suspension. Furthermore, the plating efficiency after panningwas significantly higher (up to 60%) compared to previously reportedvalues of 6 to 10%. Though the hepatoblasts still remain a minorpopulation after panning procedures, it is important to consider thatthe standard in situ hepatocyte perfusion protocols yields a populationcontaining, on average, 37.7% hepatocytes.

[0087] The advantage of this protocol in comparison with previousmethods which involved attachment of dispersed liver cells to culturedishes, low-speed differential centrifugation, and culture inarginine-deficient medium are several-fold. Isolate hepatocytes rapidlylose tissue-specific gene regulation in vitro. As a result, inprocedures requiring cell attachment to matrix, measurement ofparenchymal-specific function, such as protein or mRNA content, mightnot reflect in vivo levels. Dissociated fetal hepatoblasts also readilyform large aggregates via a calcium and temperature-dependent,glycoprotein-mediated process. As early as gestation day 14, high levelsof a cell membrane protein which is thought to be uvomorulin(E-cadherin) were present on hepatoblasts. This tendency for aggregationexplains the ability of low speed differential centrifuigation to enrichfor relatively large (E19) hepatoblasts, especially in the presence ofCa²⁺ and at temperatures greater than 4° C. To disaggregate thehepatoblasts, mechanical methods including vigorous pipetting andaspiration through a syringe have been employed but found to beinsufficient, leading to difficulties with further analyses whichrequire a single cell suspension such as FACS.

[0088] The tendency of the cells to aggregate is prevented bymaintaining the cells at 4° C. and by removing calcium with EGTA,interfering with CAM-mediated aggregation. The advantage of maintainingthe cells as a single cell suspension is two-fold. First, measurement ofparenchymal specific functions can be determined on a cellular basis,overcoming the physiologically irrelevant changes in hemopoietic cellpopulation. Second, procedures such as FACS which demand a single cellsuspension can be easily performed.

[0089] Though gestation day 15 hepatoblasts appear larger than thenon-parenchymal cells, side scatter rather than forward scatter on theFACS proved to be a better discriminator in separating the variouspopulations, presumably because even gestation day 12 hepatoblasts,which contain vacuoles, mitochondria and abundant endoplasmic reticulum,are relatively complex. In addition, side scatter proved a reasonablemeasure of cellular maturity. In general, hepatoblasts of greatergranularity were more mature morphologically and biochemically (data notshown).

[0090] Hence, FACS analysis was employed to examine the expression ofthe oval cell marker, OC.3, which has been proposed to identify liverstem cells. With multiparametric FACS analysis for OC.3 or OX-43/44expression in combination with gating for cells of particular levels ofgranularity, the inventors were able to subdivide the populations intonon-parenchymal cells (hemopoietic, endothelial, and stromal cells)versus parenchymal cell precursors that were AFP⁺. Moreover, theinventors were able to evaluate the expression of the OC.3 antigen inthe various subpopulations. At gestation day 15, most agranular,OX43/44⁺ cells proved to be hemopoietic cells, largely erythroid cellpopulations. Of the granular, OX43/44⁻ cell population, which werepredominantly AFP⁺, approximately 30% of the cells were OC.3⁺ andprobably represented bile duct cell precursors, whereas the OC.3⁻ cellswere probable hepatocyte precursors. However, a small percentage ofagranular, OX43/44⁻ cells were AFP⁺.

[0091] In comparison to the hemopoietic field, the liver stem cell fieldis still in its infancy. However, the ability to isolate specificpopulations by FACS sorting using these parameters with subsequent invitro and in vivo fate studies will greatly aid in identifying the liverstem cell. Furthermore, this technology is applicable to the study ofall aspects of liver stem cell biology including the biliary epithelium,carcinogenesis, regeneration, aging and tissue-specific gene expression.

[0092] Although the invention herein has been described with referenceto particular embodiments, it is to be understood that these embodimentsare merely illustrative of various aspects of the invention. Thus, it isto be understood that numerous modifications may be made in theillustrative embodiments and other arrangements may be devised withoutdeparting from the spirit and scope of the invention.

We claim:
 1. A method of isolating hepatoblasts from embryonic orneonatal liver comprising: (a) preparing a single cell suspension ofembryonic or neonatal liver cells; (b) panning said suspension utilizingantibodies specific for hemopoietic cells, including red blood cells,endothelial cells or other mesenchymal cells so as to remove hemopoieticcells, including red blood cells, endothelial cells and othermesenchymal cells from said suspension; and (c) performing fluorescenceactivated cell sorting utilizing said antibodies so as to removehemopoietic cells, including red blood cells, including red blood cells,endothelial cells and other mesenchymal cells from said suspension andperforming multiparametric fluorescence activated cell sorting on saidsuspension utilizing at least one antibody to a hepatic cell marker,side scatter, forward scatter and/or autofluorescence such that thecells remaining in said suspension are isolated hepatob lasts.
 2. Themethod of claim 1 wherein the antibody-specific for hemopoietic cells isa monoclonal antibody.
 3. The method of claim 2 wherein said monoclonalantibody is OX-43 and/or OX-44.
 4. The method of claim 1 wherein theantibody to a hepatic cell marker is monoclonal antibody 374.3.
 5. Themethod of claim 1 wherein said hepatic cell marker is OC.3.
 6. Themethod of claim 1 wherein said single cell suspension contains an agentcapable of removing calcium from liver cell surface.
 7. The method ofclaim 1 wherein said single cell suspension contains EGTA.
 8. The methodof claim 1 wherein said single cell suspension contains an enzymecapable of dissociating liver cells.
 9. The method of claim 1 whereinsaid single cell suspension contains collagenase.
 10. The method ofclaim 1 wherein said single cell suspension is chilled.
 11. The methodof claim 1 wherein said single cell suspension is at a temperature ofbetween about 2 and 20° C.
 12. Hepatoblasts isolated by the method ofclaim
 1. 13. A method of isolating hepatoblasts from adult livercomprising: (a) preparing a single cell suspension of adult. livercells; (b) panning said suspension utilizing antibodies specific formature hepatocytes, mature bile duct cells, endothelial cells andmesenchymal cells so as to remove mature hepatocytes, mature bile ductcells, endothelial cells and mesenchymal cells from said suspension; and(c) performing fluorescence activated cell sorting utilizing saidantibodies so as to remove mature hepatocytes, mature bile duct cells,endothelial cells and mesenchymal cells from said suspension andperforming multiparametric fluorescence activated cell sorting on saidsuspension utilizing antibody to a hepatic cell marker, side scatter,forward scatter and/or autofluorescence such that the cells remaining insaid suspension are isolated hepatoblasts.
 14. The method of claim 13wherein the antibody to a hepatic cell marker is monoclonal antibody374.3.
 15. The method of claim 13 wherein the hepatic cell marker isOC.3.
 16. The method of claim 13 wherein the single cell suspensioncontains an agent capable of removing calcium from the surface of livercells.
 17. The method of claim 13 wherein the single cell suspensioncontains EGTA.
 18. The method of claim 13 wherein the single cellsuspension contains an enzyme capable of dissociating adult liver cells.19. The method of claim 13 wherein the single cell suspension containscollagenase.
 20. The method of claim 13 where in the single cellsuspension is chilled.
 21. The method of claim 13 wherein the singlecell suspension is at a temperature of between about 2 and 20° C. 22.Hepatocytes isolated by the method of claim
 13. 23. A method of treatingliver dysfunction comprising the administration of hepatoblasts.
 24. Themethod of claim 23 wherein the administration comprises injecting saidhepatoblasts into the liver via a vascular vessel.
 25. The method ofclaim 23 wherein the administration of comprises injecting saidhepatoblasts into an ectopic site.
 26. The method of claim 23 whereinthe administration comprises injecting said hepatoblasts into an ectopicsite of the spleen.
 27. The method of claim 23 wherein the hepatoblastsare isolated by the method of claim
 1. 28. The method of claim 23wherein the hepatoblasts are isolated by the method of claim
 13. 29. Amethod of forming an artificial liver comprising the utilization ofhepatoblasts with a bioreactor.
 30. The method of claim 29 wherein thehepatoblasts are isolated by the method of claim
 1. 31. The method ofclaim 29 wherein the hepatoblasts are isolated by the method of claim13.
 32. A method of forming an artificial liver comprising theutilization of hepatoblasts in a culture apparatus.
 33. The method ofclaim 32 wherein the hepatoblasts are isolated by the method of claim 1.34. The method of claim 32 wherein the hepatoblasts are isolated by themethod of claim 13.